Kim Keehyuk, Plapp Bryce V
Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA.
Chem Biol Interact. 2017 Oct 1;276:77-87. doi: 10.1016/j.cbi.2016.12.016. Epub 2016 Dec 23.
The substrate specificities of alcohol dehydrogenases (ADH) are of continuing interest for understanding the physiological functions of these enzymes. Ser-48 and Phe-93 have been identified as important residues in the substrate binding sites of ADHs, but more comprehensive structural and kinetic studies are required. The S48T substitution in horse ADH1E has small effects on kinetic constants and catalytic efficiency (V/K) with ethanol, but decreases activity with benzyl alcohol and affinity for 2,2,2-trifluoroethanol (TFE) and 2,3,4,5,6-pentafluorobenzyl alcohol (PFB). Nevertheless, atomic resolution crystal structures of the S48T enzyme complexed with NAD and TFE or PFB are very similar to the structures for the wild-type enzyme. (The S48A substitution greatly diminishes catalytic activity.) The F93A substitution significantly decreases catalytic efficiency (V/K) for ethanol and acetaldehyde while increasing activity for larger secondary alcohols and the enantioselectivity for the R-isomer relative to the S-isomer of 2-alcohols. The doubly substituted S48T/F93A enzyme has kinetic constants for primary and secondary alcohols similar to those for the F93A enzyme, but the effect of the S48T substitution is to decrease V/K for (S)-2-alcohols without changing V/K for (R)-2-alcohols. Thus, the S48T/F93A substitutions invert the enantioselectivity for alcohol oxidation, increasing the R/S ratio by 10, 590, and 200-fold for 2-butanol, 2-octanol, and sec-phenethyl alcohol, respectively. Transient kinetic studies and simulations of the ordered bi bi mechanism for the oxidation of the 2-butanols by the S48T/F93A ADH show that the rate of hydride transfer is increased about 7-fold for both isomers (relative to wild-type enzyme) and that the inversion of enantioselectivity is due to more productive binding for (R)-2-butanol than for (S)-2-butanol in the ternary complex. Molecular modeling suggests that both of the sec-phenethyl alcohols could bind to the enzyme and that dynamics must affect the rates of catalysis.
醇脱氢酶（ADH）的底物特异性一直是理解这些酶生理功能的研究热点。Ser-48和Phe-93已被确定为ADH底物结合位点中的重要残基，但仍需要更全面的结构和动力学研究。马ADH1E中的S48T替换对乙醇的动力学常数和催化效率（V/K）影响较小，但降低了对苄醇的活性以及对2,2,2-三氟乙醇（TFE）和2,3,4,5,6-五氟苄醇（PFB）的亲和力。然而，与NAD以及TFE或PFB复合的S48T酶的原子分辨率晶体结构与野生型酶的结构非常相似。（S48A替换会极大地降低催化活性。）F93A替换显著降低了对乙醇和乙醛的催化效率（V/K），同时提高了对较大仲醇的活性以及相对于2-醇的S-异构体对R-异构体的对映选择性。双取代的S48T/F93A酶对伯醇和仲醇的动力学常数与F93A酶相似，但S48T替换的作用是降低（S）-2-醇的V/K，而不改变（R）-2-醇的V/K。因此，S48T/F93A替换使醇氧化的对映选择性发生反转，对于2-丁醇、2-辛醇和仲苯乙醇，R/S比值分别增加了10倍、590倍和200倍。对S48T/F93A ADH氧化2-丁醇的有序双底物双产物机制的瞬态动力学研究和模拟表明，两种异构体的氢化物转移速率均提高了约7倍（相对于野生型酶），并且对映选择性的反转是由于在三元复合物中（R）-2-丁醇比（S）-2-丁醇的结合更有效。分子模拟表明，两种仲苯乙醇都可以与酶结合，并且动力学必定会影响催化速率。
